BENZO[A]PYRENE INHIBITS PROTEIN-KINASE-C ACTIVITY IN SUBCULTURED RAT AORTIC SMOOTH-MUSCLE CELLS

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) interferes with protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins in vivo. To evaluate the biochemical basis of this response, the present studies have been conducted to examine the time- and concentration-dependent effects of BaP on PKC activity in vitro. Growth-arrested subcultures of rat aortic SMCs were exposed to 0.3, 3, or 30 mu M BaP in the presence of fetal bovine serum for various times and then processed for measurements of exogenous histone Type III-S phosphorylation under PKC-activating conditions. Challenge of SMCs with BaP for 8 h was associated with a concentration-dependent inhibition of PKC activity in both cytosolic and particulate fractions. While no changes of enzymatic activity were observed in either fraction following exposure of SMCs to 0.3 mu M BaP, higher concentrations of BaP inhibited PKC in both cytosolic and particulate fractions. A 49% and 68% reduction of cytosolic PKC activity was observed in SMCs treated with 3 and 30 mu M BaP, respectively. The inhibitory response elicited by BaP was more pronounced in the particulate fraction where 61% and 89% decreases in PKC activity were observed in cultures treated with 3 and 30 mu M BaP, respectively. Time course studies revealed that inhibition of PKC activity by 30 mu M BaP occurred as early as 30 min and was sustained for up to 24 h in both fractions. Benzo[a]pyrene (30 mu M) did not interfere with the ability of phorbol-12-myristate-13-acetate to induce PKC translocation from the cytosolic to particulate compartment since maximal translocation occurred by 5 min and lasted for up to 60 min in both control and BaP-treated cultures. The inhibitory effects of BaP were independent of new protein or RNA synthesis, but appear to involve oxidative metabolism of the parent compound since 3-hydroxy-BaP, the major P450-derived BaP metabolite in SMCs, also inhibited cytosolic and particulate PKC activity. Collectively, these data demonstrate that BaP and its 3-hydroxy metabolite inhibit PKC activity in rat aortic SMCs and raise the possibility that interference with PKC-mediated protein phosphorylation participates in the deregulation of SMC growth and differentiation induced by BaP.